1. Field of the Invention
This invention relates to a novel class of peptide ketoamides useful for selectively inhibiting serine proteases, selectively inhibiting cysteine proteases, generally inhibiting all serine proteases, and generally inhibiting all cysteine proteases. Serine proteases and cysteine proteases are involved in numerous disease states and inhibitors for these enzymes can be used therapeutically for the treatment of diseases involving serine proteases or cysteine proteases. We have discovered that peptide .alpha.-ketoamides can be constructed to inhibit selectively individual serine or cysteine proteases or groups of serine or cysteine proteases. We have found that peptide ketoamides which contain hydrophobic aromatic amino acid residues in the P.sub.1 site are potent inhibitors of chymases and chymotrypsin-like enzymes. Ketoamides containing small hydrophobic amino acid residues at the P.sub.1 position are good inhibitors of elastases. Inhibitors of elastases and chymases are useful as anti-inflammatory agents. We show that peptide ketoamides which contain cationic amino acid residues such as Arg and Lys in the P.sub.1 site will be potent inhibitors of trypsin and blood coagulation enzymes. These inhibitors are thus useful as anticoagulants. Cysteine proteases such as papain, cathepsin B, and calpain I and II are also inhibited by ketoamides. Ketoamides with aromatic amino acid residues in the P.sub.1 site are good inhibitors for cathepsin B and papain. Thus, they would have utility as anticancer agents. Ketoamides with either aromatic amino acid residues or small hydrophobic alkyl amino acid residues at P.sub.1 are good inhibitors of calpain I and II. These inhibitors are useful as neuroprotectants and can be used as therapeutics for the treatment of neurodegeneration and stroke.
2. Nomenclature
In discussing the interactions of peptides with serine and cysteine proteases, we have utilized the nomenclature of Schechter and Berger [Biochem. Biophys. Res. Commun. 27, 157-162 (1967); incorporated herein by reference]. The individual amino acid residues of a substrate or inhibitor are designated P.sub.1, P.sub.2, etc. and the corresponding subsites of the enzyme are designated S.sub.1, S.sub.2, etc. The scissile bond of the substrate is S.sub.1 --S.sub.1 '. The primary substrate recognition site of serine proteases is S.sub.1. The most important recognition subsites of cysteine proteases are S.sub.1 and S.sub.2.
Amino acid residues and blocking groups are designated using standard abbreviations [see J. Biol. Chem. 260, 14-42 (1985) for nomenclature rules; incorporated herein by reference]. An amino acid residue (AA) in a peptide or inhibitor structure refers to the part structure --NH--CHR.sub.1 --CO--, where R.sub.1 is the side chain of the amino acid residue AA. A peptide .alpha.-ketoester residue would be designated -AA-CO--OR which represents the part structure --NH--CHR.sub.1 --CO--CO--OR. Thus, the ethyl ketoester derived from benzoyl alanine would be designated Bz-Ala-CO-OEt which represents C.sub.6 H.sub.5 CO--NH--CHMe--CO--CO-OEt. Peptide ketoamide residues would be designated -AA-CO--NH-R. Thus, the ethyl keto amide derived from Z-Leu-Phe-OH would be designated Z-Leu-Phe-CO--NH-Et which represents C.sub.6 H.sub.5 CH.sub.2 OCO--NH--CH(CH.sub.2 CH(CH.sub.2)--CO--NH--CH(CH.sub.2 Ph)--CO--CO--NH--Et.
3. Description of the Related Art
Cysteine Proteases. Cysteine proteases such as calpain use a cysteine residue in their catalytic mechanism in contrast to serine proteases which utilize a serine residue. Cysteine proteases include papain, cathepsin B, calpains, and several viral enzymes. Neural tissues, including brain, are known to possess a large variety of proteases, including at least two calcium stimulated proteases termed calpains. Calpains are present in many tissues in addition to the brain. Calpain I is activated by micromolar concentrations of calcium while calpain II is activated by millimolar concentrations. In the brain, calpain II is the predominant form, but calpain I is found at synaptic endings and is thought to be the form involved in long term potentiation, synaptic plasticity, and cell death. Other Ca.sup.2+ activated cysteine proteases may exist, and the term "calpain" is used to refer to all Ca.sup.2+ activated cysteine proteases, including calpain I and calpain II. The terms "calpain I" and "calpain II" are used herein to refer to the micromolar and millimolar activated calpains, respectively, as described above. While calpains degrade a wide variety of protein substrates, cytoskeletal proteins seem to be particularly susceptible to attack. In some cases, the products of the proteolytic digestion of these proteins by calpain are distinctive and persistent over time. Since cytoskeletal proteins are major components of certain types of cells, this provides a simple method of detecting calpain activity in cells and tissues. Thus, calpain activation can be measured indirectly by assaying the proteolysis of the cytoskeletal protein spectrin, which produces a large, distinctive and biologically persistent breakdown product when attacked by calpain [Siman, Baudry, and Lynch, Proc. Natl. Acad. Sci. USA 81, 3572-3576 (1984); incorporated herein by reference]. Activation of calpains and/or accumulation of breakdown products of cytoskeletal elements has been observed in neural tissues of mammals exposed to a wide variety of neurodegenerative diseases and conditions. For example, these phenomena have been observed following ischemia in gerbils and rats, following stroke in humans, following administration of the toxins kainate, trimethyltin or colchicine in rats, and in human Alzheimer's disease.
Several inhibitors of calpain have been described including peptide aldehydes such as Ac-Leu-Leu-Nle-H and Ieupeptin (Ac-Leu-Leu-Arg-H), as well as epoxysuccinates such as E-64. These compounds are not especially useful at inhibiting calpain in neural tissue in vivo because they are poorly membrane permeant and, accordingly, are not likely to cross the blood brain barrier very well. Also, many of these inhibitors have poor specificity and will inhibit a wide variety of proteases in addition to calpain. Other classes of compounds which inhibit cysteine proteases include peptide diazomethyl ketone [Rich, D. H., in Protease Inhibitors, Barrett A. J., and Salversen, G., Eds., Elsevier, New York, 1986, pp 153-178; incorporated herein by reference). Peptide diazomethyl ketones are potentially carcinogenic and are thought to be poorly membrane permeant and to have low specificity. Thus, no effective therapy has yet been developed for most neurodegenerative diseases and conditions. Millions of individuals suffer from neurodegenerative diseases and thus, there is a need for therapies effective in treating and preventing these diseases and conditions.
Cathepsin B is involved in muscular dystrophy, myocardial tissue damage, tumor metastasis, and bone resorption. In addition, a number of viral processing enzymes, which are essential for viral infection, are cysteine proteases. Inhibitors of cysteine proteases would have multiple therapeutic uses.
Serine Proteases. Serine proteases play critical roles in several physiological processes such as digestion, blood coagulation, complement activation, fibrinolysis, viral infection, fertilization, and reproduction. Serine proteases are not only a physiological necessity, but also a potential hazard if they are not controlled. Uncontrolled proteolysis by elastases may cause pancreatitis, emphysema, rheumatoid arthritis, bronchial inflammation and adult respiratory distress syndrome. It has been suggested that a new trypsin-like cellular enzyme (tryptase) is involved in the infection of human immunodeficiency virus type 1 [HIV-1; Hattori et al., FEBS Letters 248, pp. 48-52 (1989)], which is a causative agent of acquired immunodeficiency syndrome (AIDS). Plasmin is involved in tumor invasiveness, tissue remodeling, blistering, and clot dissociation. Accordingly, specific and selective inhibitors of these proteases should be potent anticoagulants, anti-inflammatory agents, anti-tumor agents and anti-viral agents useful in the treatment of protease-related diseases [Powers and Harper, Proteinase Inhibitors, pp 55-152, Barrett and Salvesen, eds., Elsevier, (1986); incorporated herein by reference]. In vitro proteolysis by chymotrypsin, trypsin or the elastase family is a serious problem in the production, purification, isolation, transport or storage of peptides and proteins.
Elastase inhibitors are anti-inflammatory agents which can be used to treat elastase-associated inflammation including rheumatoid arthritis and emphysema. Although the naturally occurring protease inhibitor, .alpha.1-protease inhibitor (.alpha.1-PI) has been used to treat patients with emphysema, this protein inhibitor is not widely used clinically due to the high dosage needed for treatment and the difficulty of producing large quantifies. Therefore, small molecular weight elastase inhibitors are needed for therapy. Other low molecular weight elastase inhibitors have utility for the treatment of emphysema and intimation (see: 1-carpapenem-3-carboxylic esters as anti-inflammatory agents, U.S. Pat. No. 4,493,839; N-carboxyl-thienamycin esters and analogs thereof as anti-inflammatory agents, U.S. Pat. No. 4,495,197; incorporated herein by reference).
Anticoagulants and antithrombotic drugs are used in a variety of thrombotic disorders. The 1990 Physician's Desk Reference lists several anticoagulant drugs (heparin, protamine sulfate and warfarin), a few antiplatelet drugs (aspirin) and several thrombolytic agents. Heparin and warfarin are commonly used clinically for prevention and treatment of venous thrombosis and pulmonary embolism. Heparin inhibits the blood coagulation activity by accelerating the binding of natural plasma protease inhibitor antithrombin III with coagulation factors, and warfarin acts as a vitamin K antagonist and inhibits the synthesis of coagulation factors. None of the anticoagulant drugs, antithrombotic drugs, fibrinolytic agents and antiplatelet drugs are highly effective in all clinical situations and many induce side reactions [Von Kaulla, Burger's Medicinal Chemistry, Part II, pp 1081-1132, Wolff, ed., (1979); incorporated herein by reference]. Coagulation disorders such as disseminated intravascular coagulation, bleeding complications of medical and surgical procedures and bleeding complications of systemic illness are still difficult to manage [Ingram, Brozovic and Slater, Bleeding Disorders, pp 1-413, Blackwell Scientific Publications, (1982); incorporated herein by reference]. In the treatment of patients with coagulation problems, anticoagulant or antithrombotic agents of diverse mechanisms are urgently sought in order to provide better medical care. Inhibitors for the trypsin-like enzymes involved in blood coagulation are useful anticoagulants in vivo [see for example: H-D-Phe-Pro-Arg-CH.sub.2 C.sub.1, Hanson and Harker, Proc. Natl. Acad. Sci. 85, 3184-3188 (1988); 7-Amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin (ACITIC), Oweida, Ku, Luresden, Karo, and Powers, Thrombos. Res. 58, 191-197 (1990); incorporated herein by reference].
Ketoesters. A few amino acid and peptide ketoesters and ketoacids have been previously reported. Cornforth and Cornforth [J. Chem. Soc., 93-96 (1953); incorporated herein by reference] report the synthesis of the ketoacids PhCH.sub.2 CO-Gly-CO--OH and Ac-Gly-CO--OH upon hydrolysis of heterocyclic molecules. Charles et al. [J. Chem. Soc. Perkin I, 1139-1146 (1980); incorporated herein by reference] use ketoesters for the synthesis of bicyclic heterocycles. They report the synthesis of n-BuCO-Ala-CO-OEt, PrCO-Ala-CO-OEt, cyclopentylCO-Ala-CO-OEt, PrCO-PhGly-CO-OEt, and Bz-Ala-CO-OEt. Hori et al. [Peptides: Structure and Function-Proceedings of the Ninth American Peptide Symposium (Deber, Hruby, and Kopple, Eds.) Pierce Chemical Co., pp 819-822 (1985); incorporated herein by reference] report Bz-Ala-CO-OEt, Bz-Ala-CO--OH, Z-Ala-Ala-Abu-CO-OEt, Z-Ala-Ala-Abu-CO-OBzl, and Z-Ala-Ala-Ala-Ala-CO-OEt (Abu=2-aminobulanoic acid or aminobutyric acid) and report that these compounds inhibit elastase. Trainer [Trends Pharm. Sci. 8, 303-307 (1987); incorporated herein by reference] comments on one of this compounds. Burkhart, J., Peet, N. P., and Bey, P. [Tetrahedron Lett. 29, 3433-3436 (1988); incorporated herein by reference] report the synthesis of Z-Val-Phe-CO-OMe and Bz-Phe-CO-OMe.
Mehdi et al., [Biochem. Biophys. Res. Comm. 166, 595-600 (1990); incorporated herein by reference] report the inhibition of human neutrophil elastase and cathepsin G by peptide .alpha.-ketoesters. Angelastro et al., [J. Med. Chem. 33, 13-16 (1990); incorporated herein by reference] report some .alpha.-ketoesters which are inhibitors of calpain and chymotrypsin. Hu and Abeles [Arch. Biochem. Biophys. 281, 271-274 (1990)]; incorporated herein by reference] report some peptidyl .alpha.-ketoamides and .alpha.-ketoacids which are inhibitors of cathepsin B and papain. Peet et al. [J. Med. Chem. 33, 394-407 (1990); incorporated herein by reference] report some peptidyl .alpha.-ketoesters which are inhibitors of porcine pancreatic elastase, human neutrophil elastase, and rat & human neutrophil cathepsin G.
Ketoamides. A single peptide ketoamide is reported in the literature by Hu and Abeles [Arch. Biochem. Biophys. 281, 271-274 (1990)]. This compound Z-Phe-NHCH.sub.2 CO--CO--NH-Et or Z-Phe-Gly-CO--NH-Et is reported to be an inhibitor of papain (K.sub.I =1.5 .mu.M) and cathepsin B (K.sub.I =4 .mu.M).